The Tox21 programs federal partners include the Environmental Protection Agency (EPA), the Food and Drug Administration (FDA) and NIH, with leadership from NCATS and the National Toxicology Program (NTP) at the National Institute of Environmental Health Sciences (NIEHS). These agencies work together to advance in vitro toxicological testing. The Tox21 Program is comprised of three NCATS teams: Systems Toxicology, Genomic Toxicology, and Computational Toxicology. The Systems Toxicology team has identified, developed, optimized, and/or screened more than 19 assays. Highlights range from performing 10 online screens, including estrogen-related receptor (ERR)/proliferator-activated-receptor-gamma coactivator (ERR/PGC), thyroid stimulating hormone receptor (TSHR) and histone deacetylase I/II (HDAC I/II) assays in triplicate against the Tox21 10K compound collection and 9 online validation assays, including cell-based acetylcholinesterase (AChE) assay, signaling pathway and nuclear receptor assays, against the LOPAC collection on the Tox21 robotic system. All of these assays were optimized and evaluated before moving to robotic online validation and online screening. AChE is the primary cholinesterase in the body that metabolizes a key neurotransmitter, acetylcholine. Inhibition of AChE activity can lead to neurotoxicity and known inhibitors include organophosphorus pesticides, chemical warfare agents, drugs, and various phytochemicals. In collaboration with the CFSAN/FDA, the Systems Toxicology team has developed two cell-based AChE assays with fluorescence and color metric readouts in a homogenous format and validated these assays by screening the LOPAC library. Androgen receptor (AR), a nuclear receptor, is an important transcription factor regulating gene expression for male sexual development and maintaining accessory sexual organ function. Disruption of AR by environmental chemicals has many possible adverse outcomes in humans including multiple types of endocrine disruption such as changes in spermatogenesis and the synthesis of sex hormones. The team has identified a group of potential androgen receptor disruptors from the Tox21 10K screening. Several follow-up studies have been performed, including a biochemical assay determining the binding affinity, as well as a translocation assay to identify AR translocation into the nucleus. In addition, the team has worked on the mitochondria project and selected about 800 compounds to be tested in rat hepatocyte MMP, reactive oxygen species (ROS), and ATP content assays. About 50 compounds were further tested in pathway assays, C. elegans models, mitochondrial gene expression experiment using RASL-seq, and oxygen consumption assays. In collaboration with NIEHS, we optimized and screened PGC-ERR and ERR luciferase reporter gene assays against Tox21 10K compound collection. After the primary screening, 1000 compounds were cherry picked and re-tested in the ERR and PGC-ERR assays. The team has also searched for epigenetic assays for the Tox21 Program by optimizing a cell-based HDAC I/II assay in various cancer cell and stem cell backgrounds and validating this assay by screening the NCATS Pharmaceutical Collection of 2,816 approved and investigational drugs. The compounds were also tested with a control assay to identify false positives as results of luciferase inhibition or protease inhibition. Using an orthogonal HDAC biochemical assay, we profiled compound selectivity of 30 identified HDAC inhibitors against a panel of HDAC subtypes. The TRHR project is a collaborative effort with the EPA to generate and evaluate the TRHR cell line by measuring calcium levels after TRH stimulation. This assay has been validated online and is ready for online screening. In addition, the team has identified a group of CAR activators and deactivators from the primary screening. Several follow-up studies have been performed to confirm the compounds with CAR activity.